Frequently Asked Questions

General

If you would like to place an order, please request a quote online. If you are sequencing an organism from our white list, we will send you a barcoded bead tube for each strain. Alternatively, you can send us extracted DNA (in which case we will send you barcodes only). It is your responsibility to put the correct strain in each tube and return them to us. The tubes will be loaded onto our robot to perform DNA extraction and Illumina library preparation, and they will then be sequenced on an Illumina HiSeq or MiSeq. The raw sequence data will be processed using our automated analysis pipeline, and returned to you via a website.

We are funded by the BBSRC until 2019, and this effectively covers our staffing and equipment costs, so we are able to offer our service to BBSRC-funded researchers on a consumables-only basis. Additionally, our costs are minimised due to extensive automation and economies of scale.

Currently, we charge £50 for BBSRC-funded projects, £70 for other non-commercial projects, and £100 for industrial projects. These prices are excluding VAT.

We aim to make the analysed sequence data available to you within eight to ten weeks of receiving the sample. You can see our live median wait time on the home page.

No, MicrobesNG is a BBSRC-funded collaboration between the University of Birmingham and the University of Sheffield. It is an academic project, although we are happy to accept samples from industry at our industrial rates.

We do not require co-authorship for our standard pipeline analysis. However, please make sure to acknowledge our service by including the following text in the acknowledgements section:

Genome sequencing was provided by MicrobesNG (http://www.microbesng.uk), which is supported by the BBSRC (grant number BB/L024209/1).

Such acknowledgements are critical to the long-term maintenance of the service.

If your project involves additional sequencing or bioinformatics not covered by our standard pipeline, then we may be able to help on a collaborative basis. In those circumstances, co-authorship would be expected.

Strain Archive

If you choose it to be. You will also have the option of the embargo period listed below.

Our embargo period is 12 months from when we send you your data. We are not in the position to negotiate with each individual group about the embargo.

If you have strains which you do not want to be made publicly available at all, you have that option if you sequence them with us at our industrial rates of £100+VAT per sample.

No, we are not currently licensed to handle any organisms not on our white list. We can still provide you with a sequencing and analysis service, but you will have to perform the extractions yourself and send us DNA rather than the organism.

The BBSRC have funded us to set up an open, accessible community resource for researchers in the UK. As such it is a requirement for us to make the strains and sequence data publicly available. An embargo period can be put in place allowing you time to publish before the information becomes public.

Our embargo period is 12 months from when we send you your data. We are not in the position to negotiate with each individual group about the embargo.

If you have strains which you do not want to be made publicly available at all, you have that option if you sequence them with us at our industrial rates of £100+VAT per sample.

Currently, we are not able to accept strains from outside the UK. If you want to sequence strains with us from outside UK you can send us genomic DNA.

When preparing DNA extractions for sequencing with us, make sure they are eluted in EB buffer (i.e. 10 mM Tris-HCl pH 8.5) with no EDTA. Do not use TE for eluting the DNA as it contains EDTA,which inhibits our NGS library preparation. In the absence of a buffering agent, store samples at -25°C to -15°C to prevent degradation.

Sequencing

Currently, all sequencing is performed on the Illumina MiSeq and HiSeq 2500 platforms.

All projects will be sequenced using 2x250bp paired-end reads, regardless of whether the MiSeq or HiSeq is used.

There are no plans to obtain a Pacific Biosciences sequencer in Birmingham. PacBio sequencing as a service is available in the UK via the Centre for Genomic Research at the University of Liverpool, and also by collaboration with the Wellcome Trust Sanger Institute or TGAC.

We are involved in the Oxford Nanopore MinION Access Programme (MAP), and Nick Loman is at the forefront of this development, having published one of the first software packages for analysis of Oxford Nanopore sequence data (poretools), and the first complete genome assembly obtained entirely using Oxford Nanopore data. Oxford Nanopore sequencing is not yet commercially available, but once it is released, it is likely that we will add it as an option for MicrobesNG projects (at additional cost).

No, currently MicrobesNG is focused on providing draft sequencing of microbial genomes as a sole application. However, in Birmingham we have performed the sequencing for projects using all of those methods, and may be interested in working on such projects on a collaborative basis outside of MicrobesNG. It should be noted that all of these methods typically require considerably greater sequencing depth than genome sequencing, so would be proportionally more expensive.

No, microbial genomes are much more interesting.

Bioinformatics

All projects are put through a standard analysis pipeline. We identify the closest available reference genome using Kraken, and map the reads to this using BWA mem to assess the quality of the data. We also perform a de novo assembly of the reads using SPAdes, and map the reads back to the resultant contigs, again using BWA mem to get more quality metrics.

Upon receipt of a suitable reference (supplied by the user) we can predict variants relative to the reference. Variant calling is performed using VarScan. With the same reference contigs will be reordered and reoriented relative to the reference genome based on a MUMmer whole-genome alignment, and an automated annotation will be performed using Prokka. All data will be provided to you via a user-friendly web interface.

The MicrobesNG pipeline produces draft genome sequences, so you will not get back a closed genome. Repeat sequences larger than ~1000bp (e.g. IS elements and rRNA operons) cannot be resolved using our sequencing methods, and will cause a break in the assembly.

The number of contigs you will receive depends on the repeat structure of the genome. For an E. coli strain we would typically expect to obtain fewer than 200 contigs, with an n50 of >100kb (the n50 value means that at least half the genome is assembled into contigs of that size or greater).

The outputs from the analysis pipeline are provided using standard bioinformatics file formats including fasta, gbk, gff, fastq and finally, upon receipt of a reference genome from the customer, vcf and bam.

No, all samples are put through the same bioinformatics pipeline, which forms part of our QC procedure, and is provided at no additional cost. However, you will be able to download the raw sequence data in Fastq format, suitable for use with most downstream analysis tools (including BWA, Bowtie2, SPAdes and CLCBio).

MicrobesNG does not provide a bespoke analysis service, just the automated pipeline. However, we may be interested in helping on a collaborative basis.